Integration of biological data by kernels on graph nodes allows prediction of new genes involved in mitotic chromosome condensation.
Heriche, J.K., Lees, J.G., Morilla, I., Walter, T., Petrova, B., Julia Roberti, M., Hossain, M.J., Adler, P., Fernandez, J.M., Krallinger, M., Haering, C.H., Vilo, J., Valencia, A., Ranea, J.A., Orengo, C. & Ellenberg, J.
Mol Biol Cell. 2014 Jun 18. pii: mbc.E13-04-0221.
The advent of genome-wide RNAi-based screens puts us in the position to identify genes for all functions human cells carry out. However, for many functions, assay complexity and cost make genome-scale knockdown experiments impossible. Methods to predict genes required for cell functions are therefore needed to focus RNAi screens from the whole genome to the most likely candidates. While different bioinformatics tools for gene function prediction exist, they lack experimental validation and are therefore rarely used by experimentalists. To address this, we developed an effective computational gene selection strategy that represents public data about genes as graphs and then analyses these graphs using kernels on graph nodes to predict functional relationships. To demonstrate its performance, we predicted human genes required for a poorly understood cellular function, mitotic chromosome condensation, and experimentally validated the top 100 candidates with a focused RNAi screen by automated microscopy. Quantitative analysis of the images demonstrated that the candidates were indeed strongly enriched in condensation genes including the discovery of several new factors. By combining bioinformatics prediction with experimental validation, our study shows that kernels on graph nodes are a powerful tool to integrate public biological data and predict genes involved in cellular functions of interest.
Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits.
Piazza, I., Rutkowska, A., Ori, A., Walczak, M., Metz, J., Pelechano, V., Beck, M. & Haering, C.H.
Nat Struct Mol Biol. 2014 May 18. doi: 10.1038/nsmb.2831.
Condensin complexes have central roles in the three-dimensional organization of chromosomes during cell divisions, but how they interact with chromatin to promote chromosome segregation is largely unknown. Previous work has suggested that condensin, in addition to encircling chromatin fibers topologically within the ring-shaped structure formed by its SMC and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA formed by the two HEAT-repeat subunits of the Saccharomyces cerevisiae condensin complex. From detailed mapping data of the interfaces between the HEAT-repeat and kleisin subunits, we generated condensin complexes that lack one of the HEAT-repeat subunits and consequently fail to associate with chromosomes in yeast and human cells. The finding that DNA binding by condensin's HEAT-repeat subunits stimulates the SMC ATPase activity suggests a multistep mechanism for the loading of condensin onto chromosomes.
Entrapment of chromosomes by condensin rings prevents their breakage during cytokinesis.
Cuylen, S., Metz, J., Hruby, A. & Haering, C.H.
Dev Cell. 2013 Nov 25;27(4):469-78. doi: 10.1016/j.devcel.2013.10.018.
Successful segregation of chromosomes during mitosis and meiosis depends on the action of the ring-shaped condensin complex, but how condensin ensures the complete disjunction of sister chromatids is unknown. We show that the failure to segregate chromosome arms, which results from condensin release from chromosomes by proteolytic cleavage of its ring structure, leads to a DNA damage checkpoint-dependent cell-cycle arrest. Checkpoint activation is triggered by the formation of chromosome breaks during cytokinesis, which proceeds with normal timing despite the presence of lagging chromosome arms. Remarkably, enforcing condensin ring reclosure by chemically induced dimerization just before entry into anaphase is sufficient to restore chromosome arm segregation. We suggest that topological entrapment of chromosome arms by condensin rings ensures their clearance from the cleavage plane and thereby avoids their breakage during cytokinesis.
Condensin: crafting the chromosome landscape.
Piazza, I., Haering, C.H. & Rutkowska, A.
Chromosoma. 2013 Jun;122(3):175-90. doi: 10.1007/s00412-013-0405-1. Epub 2013 Apr2.
The successful transmission of complete genomes from mother to daughter cells during cell divisions requires the structural re-organization of chromosomes into individualized and compact structures that can be segregated by mitotic spindle microtubules. Multi-subunit protein complexes named condensins play a central part in this chromosome condensation process, but the mechanisms behind their actions are still poorly understood. An increasing body of evidence suggests that, in addition to their role in shaping mitotic chromosomes, condensin complexes have also important functions in directing the three-dimensional arrangement of chromatin fibers within the interphase nucleus. To fulfill their different functions in genome organization, the activity of condensin complexes and their localization on chromosomes need to be strictly controlled. In this review article, we outline the regulation of condensin function by phosphorylation and other posttranslational modifications at different stages of the cell cycle. We furthermore discuss how these regulatory mechanisms are used to control condensin binding to specific chromosome domains and present a comprehensive overview of condensin's interaction partners in these processes.
Quantitative analysis of chromosome condensation in fission yeast.
Petrova, B., Dehler, S., Kruitwagen, T., Heriche, J.K., Miura, K. & Haering, C.H.
Mol Cell Biol. 2013 Mar;33(5):984-98. doi: 10.1128/MCB.01400-12. Epub 2012 Dec21.
Chromosomes undergo extensive conformational rearrangements in preparation for their segregation during cell divisions. Insights into the molecular mechanisms behind this still poorly understood condensation process require the development of new approaches to quantitatively assess chromosome formation in vivo. In this study, we present a live-cell microscopy-based chromosome condensation assay in the fission yeast Schizosaccharomyces pombe. By automatically tracking the three-dimensional distance changes between fluorescently marked chromosome loci at high temporal and spatial resolution, we analyze chromosome condensation during mitosis and meiosis and deduct defined parameters to describe condensation dynamics. We demonstrate that this method can determine the contributions of condensin, topoisomerase II, and Aurora kinase to mitotic chromosome condensation. We furthermore show that the assay can identify proteins required for mitotic chromosome formation de novo by isolating mutants in condensin, DNA polymerase epsilon, and F-box DNA helicase I that are specifically defective in pro-/metaphase condensation. Thus, the chromosome condensation assay provides a direct and sensitive system for the discovery and characterization of components of the chromosome condensation machinery in a genetically tractable eukaryote.
Understanding chromatin and chromosomes: from static views to dynamic thinking.
Haering, C.H. & Losada, A.
EMBO Rep. 2013 Feb;14(2):109-11. doi: 10.1038/embor.2012.221. Epub 2013 Jan 15.
The 106th Boehringer Ingelheim Fonds International Titisee Conference, 'Reconstituting Chromatin: From Self-assembly to Self-organization', took place in October 2012. The organizers, Andrea Musacchio and Tom Muir, brought together biologists, chemists and physicists to discuss the principles of chromosome assembly and organization. Topics of discussion ranged from new insights gained from the static views provided by crystal structures to analyses of chromatin dynamics inside living cells.
Cohesin in determining chromosome architecture.
Haering, C.H. & Jessberger, R.
Exp Cell Res. 2012 Jul 15;318(12):1386-93. Epub 2012 Mar 24.
Cells use ring-like structured protein complexes for various tasks in DNA dynamics. The tripartite cohesin ring is particularly suited to determine chromosome architecture, for it is large and dynamic, may acquire different forms, and is involved in several distinct nuclear processes. This review focuses on cohesin's role in structuring chromosomes during mitotic and meiotic cell divisions and during interphase.
A FlAsH-Based Cross-Linker to Study Protein Interactions in Living Cells.
Rutkowska, A., Häring, C.H. & Schultz, C.
Angew Chem Int Ed Engl. 2011 Dec 23;50(52):12655-8. doi:10.1002/anie.201106404. Epub 2011 Nov 16.
As you like it: xCrAsH, a dimeric derivative of the arsenical compound FlAsH, enables the highly specific, covalent cross-linking of two proteins containing a 12 amino acid peptide tag. This inducible and (by addition of dithiols) reversible system can be used to detect and manipulate protein-protein interactions both in vitro and in living cells.
Condensin engages chromatin.
Petrova, B. & Haering, C.H.
Chembiochem. 2011 Nov 4;12(16):2399-401. doi: 10.1002/cbic.201100531. Epub 2011Sep 23. Europe PMC
Deciphering condensin action during chromosome segregation.
Cuylen, S. & Haering, C.H.
Trends Cell Biol. 2011 Sep;21(9):552-9. doi: 10.1016/j.tcb.2011.06.003. Epub 2011Jul 15.
The correct segregation of eukaryotic genomes requires the resolution of sister DNA molecules and their movement into opposite halves of the cell before cell division. The dynamic changes chromosomes need to undergo during these events depend on the action of a multi-subunit SMC (structural maintenance of chromosomes) protein complex named condensin, but its molecular function in chromosome segregation is still poorly understood. Recent studies suggest that condensin has a role in the removal of sister chromatid cohesin, in sister chromatid decatenation by topoisomerases, and in the structural reconfiguration of mitotic chromosomes. In this review we discuss possible mechanisms that could explain the variety of condensin actions during chromosome segregation.
Condensin structures chromosomal DNA through topological links.
Cuylen, S., Metz, J. & Häring, C.H.
Nat Struct Mol Biol. 2011 Jul 17;18(8):894-901. doi: 10.1038/nsmb.2087.
The multisubunit condensin complex is essential for the structural organization of eukaryotic chromosomes during their segregation by the mitotic spindle, but the mechanistic basis for its function is not understood. To address how condensin binds to and structures chromosomes, we have isolated from Saccharomyces cerevisiae cells circular minichromosomes linked to condensin. We find that either linearization of minichromosome DNA or proteolytic opening of the ring-like structure formed through the connection of the two ATPase heads of condensin's structural maintenance of chromosomes (SMC) heterodimer by its kleisin subunit eliminates their association. This suggests that condensin rings encircle chromosomal DNA. We further show that release of condensin from chromosomes by ring opening in dividing cells compromises the partitioning of chromosome regions distal to centromeres. Condensin hence forms topological links within chromatid arms that provide the arms with the structural rigidity necessary for their segregation.
A positively charged channel within the Smc1/Smc3 hinge required for sister chromatid cohesion.
Kurze, A., Michie, K.A., Dixon, S.E., Mishra, A., Itoh, T., Khalid, S., Strmecki, L., Shirahige, K., Haering, C.H., Lowe, J. & Nasmyth, K.
EMBO J. 2011 Jan 19;30(2):364-78. doi: 10.1038/emboj.2010.315. Epub 2010 Dec 7.
Cohesin's structural maintenance of chromosome 1 (Smc1) and Smc3 are rod-shaped proteins with 50-nm long intra-molecular coiled-coil arms with a heterodimerization domain at one end and an ABC-like nucleotide-binding domain (NBD) at the other. Heterodimerization creates V-shaped molecules with a hinge at their centre. Inter-connection of NBDs by Scc1 creates a tripartite ring within which, it is proposed, sister DNAs are entrapped. To investigate whether cohesin's hinge functions as a possible DNA entry gate, we solved the crystal structure of the hinge from Mus musculus, which like its bacterial counterpart is characterized by a pseudo symmetric heterodimeric torus containing a small channel that is positively charged. Mutations in yeast Smc1 and Smc3 that together neutralize the channel's charge have little effect on dimerization or association with chromosomes, but are nevertheless lethal. Our finding that neutralization reduces acetylation of Smc3, which normally occurs during replication and is essential for cohesion, suggests that the positively charged channel is involved in a major conformational change during S phase.
A new cohesive team to mediate DNA looping.
Cuylen, S. & Häring, C.H.
Cell Stem Cell. 2010 Oct 8;7(4):424-6.
To control cell-type specific gene expression, transcription factors bound at distant enhancer sites need to come into the vicinity of promoters. In a recent Nature article, Kagey et al. (2010) provide evidence that Mediator and Cohesin protein complexes cooperate in the formation of enhancer-promoter DNA loops.
Cohesin: its roles and mechanisms.
Nasmyth, K. & Häring, C.H.
Annu Rev Genet. 2009;43:525-58.
The cohesin complex is a major constituent of interphase and mitotic chromosomes. Apart from its role in mediating sister chromatid cohesion, it is also important for DNA double-strand-break repair and transcriptional control. The functions of cohesin are regulated by phosphorylation, acetylation, ATP hydrolysis, and site-specific proteolysis. Recent evidence suggests that cohesin acts as a novel topological device that traps chromosomal DNA within a large tripartite ring formed by its core subunits.
Foreword: the many fascinating functions of SMC protein complexes.
Chromosome Res. 2009;17(2):127-9. doi: 10.1007/s10577-008-9008-8. Europe PMC
The cohesin ring concatenates sister DNA molecules.
Häring, C.H., Farcas, A.M., Arumugam, P., Metson, J. & Nasmyth, K.
Nature. 2008 Jul 17;454(7202):297-301. Epub 2008 Jul 2.
Sister chromatid cohesion, which is essential for mitosis, is mediated by a multi-subunit protein complex called cohesin. Cohesin's Scc1, Smc1 and Smc3 subunits form a tripartite ring structure, and it has been proposed that cohesin holds sister DNA molecules together by trapping them inside its ring. To test this, we used site-specific crosslinking to create chemical connections at the three interfaces between the three constituent polypeptides of the ring, thereby creating covalently closed cohesin rings. As predicted by the ring entrapment model, this procedure produced dimeric DNA-cohesin structures that are resistant to protein denaturation. We conclude that cohesin rings concatenate individual sister minichromosome DNA molecules.
Cohesin's ATPase activity is stimulated by the C-terminal Winged-Helix domain of its kleisin subunit.
Arumugam, P., Nishino, T., Häring, C.H., Gruber, S. & Nasmyth, K.
Curr Biol. 2006 Oct 24;16(20):1998-2008.
BACKGROUND: Cohesin, a multisubunit protein complex conserved from yeast to humans, holds sister chromatids together from the onset of replication to their separation during anaphase. Cohesin consists of four core subunits, namely Smc1, Smc3, Scc1, and Scc3. Smc1 and Smc3 proteins are characterized by 50-nm-long anti-parallel coiled coils flanked by a globular hinge domain and an ABC-like ATPase head domain. Whereas Smc1 and Smc3 heterodimerize via their hinge domains, the kleisin subunit Scc1 connects their ATPase heads, and this results in the formation of a large ring. Biochemical studies suggest that cohesin might trap sister chromatids within its ring, and genetic evidence suggests that ATP hydrolysis is required for the stable association of cohesin with chromosomes. However, the precise role of the ATPase domains remains enigmatic. RESULTS: Characterization of cohesin's ATPase activity suggests that hydrolysis depends on the binding of ATP to both Smc1 and Smc3 heads. However, ATP hydrolysis at the two active sites is not per se cooperative. We show that the C-terminal winged-helix domain of Scc1 stimulates the ATPase activity of the Smc1/Smc3 heterodimer by promoting ATP binding to Smc1's head. In contrast, we do not detect any effect of Scc1's N-terminal domain on Smc1/Smc3 ATPase activity. CONCLUSIONS: Our studies reveal that Scc1 not only connects the Smc1 and Smc3 ATPase heads but also regulates their ATPase activity.
The structure and function of SMC and kleisin complexes.
Nasmyth, K. & Häring, C.H.
Annu Rev Biochem. 2005;74:595-648.
Protein complexes consisting of structural maintenance of chromosomes (SMC) and kleisin subunits are crucial for the faithful segregation of chromosomes during cell proliferation in prokaryotes and eukaryotes. Two of the best-studied SMC complexes are cohesin and condensin. Cohesin is required to hold sister chromatids together, which allows their bio-orientation on the mitotic spindle. Cleavage of cohesin's kleisin subunit by the separase protease then triggers the movement of sister chromatids into opposite halves of the cell during anaphase. Condensin is required to organize mitotic chromosomes into coherent structures that prevent them from getting tangled up during segregation. Here we describe the discovery of SMC complexes and discuss recent advances in determining how members of this ancient protein family may function at a mechanistic level.
Structure and stability of cohesin's Smc1-kleisin interaction.
Häring, C.H., Schoffnegger, D., Nishino, T., Helmhart, W., Nasmyth, K. & Lowe, J.
Mol Cell. 2004 Sep 24;15(6):951-64.
A multisubunit complex called cohesin forms a huge ring structure that mediates sister chromatid cohesion, possibly by entrapping sister DNAs following replication. Cohesin's kleisin subunit Scc1 completes the ring, connecting the ABC-like ATPase heads of a V-shaped Smc1/3 heterodimer. Proteolytic cleavage of Scc1 by separase triggers sister chromatid disjunction, presumably by breaking the Scc1 bridge. One half of the SMC-kleisin bridge is revealed here by a crystal structure of Smc1's ATPase complexed with Scc1's C-terminal domain. The latter forms a winged helix that binds a pair of beta strands in Smc1's ATPase head. Mutation of conserved residues within the contact interface destroys Scc1's interaction with Smc1/3 heterodimers and eliminates cohesin function. Interaction of Scc1's N terminus with Smc3 depends on prior C terminus connection with Smc1. There is little or no turnover of Smc1-Scc1 interactions within cohesin complexes in vivo because expression of noncleavable Scc1 after DNA replication does not hinder anaphase.
Building and breaking bridges between sister chromatids.
Häring, C.H. & Nasmyth, K.
Bioessays. 2003 Dec;25(12):1178-91.
Eukaryotic chromosomes undergo dramatic changes and movements during mitosis. These include the individualization and compaction of the two copies of replicated chromosomes (the sister chromatids) and their subsequent segregation to the daughter cells. Two multisubunit protein complexes termed 'cohesin' and 'condensin', both composed of SMC (Structural Maintenance of Chromosomes) and kleisin subunits, have emerged as crucial players in these processes. Cohesin is required for holding sister chromatids together whereas condensin, together with topoisomerase II, has an important role in organizing individual axes of sister chromatids prior to their segregation during anaphase. SMC and kleisin complexes also regulate the compaction and segregation of bacterial nucleoids. New research suggests that these ancient regulators of chromosome structure might function as topological devices that trap chromosomal DNA between 50 nm long coiled coils.
ATP hydrolysis is required for cohesin's association with chromosomes.
Arumugam, P., Gruber, S., Tanaka, K., Häring, C.H., Mechtler, K. & Nasmyth, K.
Curr Biol. 2003 Nov 11;13(22):1941-53.
BACKGROUND: A multi-subunit protein complex called cohesin is involved in holding sister chromatids together after DNA replication. Cohesin contains four core subunits: Smc1, Smc3, Scc1, and Scc3. Biochemical studies suggest that Smc1 and Smc3 each form 50 nm-long antiparallel coiled coils (arms) and bind to each other to form V-shaped heterodimers with globular ABC-like ATPases (created by the juxtaposition of N- and C-terminal domains) at their apices. These Smc "heads" are connected by Scc1, creating a tripartite proteinaceous ring. RESULTS: To investigate the role of Smc1 and Smc3's ATPase domains, we engineered smc1 and smc3 mutations predicted to abolish either ATP binding or hydrolysis. All mutations abolished Smc protein function. The binding of ATP to Smc1, but not Smc3, was essential for Scc1's association with Smc1/3 heterodimers. In contrast, mutations predicted to prevent hydrolysis of ATP bound to either head abolished cohesin's association with chromatin but not Scc1's ability to connect Smc1's head with that of Smc3. Inactivation of the Scc2/4 complex had a similar if not identical effect; namely, the production of tripartite cohesin rings that cannot associate with chromosomes. CONCLUSIONS: Cohesin complexes whose heads have been connected by Scc1 must hydrolyze ATP in order to associate stably with chromosomes. If chromosomal association is mediated by the topological entrapment of DNA inside cohesin's ring, then ATP hydrolysis may be responsible for creating a gate through which DNA can enter. We suggest that ATP hydrolysis drives the temporary disconnection of Scc1 from Smc heads that are needed for DNA entrapment and that this process is promoted by Scc2/4.
Chromosomal cohesin forms a ring.
Gruber, S., Häring, C.H. & Nasmyth, K.
Cell. 2003 Mar 21;112(6):765-77.
The cohesin complex is essential for sister chromatid cohesion during mitosis. Its Smc1 and Smc3 subunits are rod-shaped molecules with globular ABC-like ATPases at one end and dimerization domains at the other connected by long coiled coils. Smc1 and Smc3 associate to form V-shaped heterodimers. Their ATPase heads are thought to be bridged by a third subunit, Scc1, creating a huge triangular ring that could trap sister DNA molecules. We address here whether cohesin forms such rings in vivo. Proteolytic cleavage of Scc1 by separase at the onset of anaphase triggers its dissociation from chromosomes. We show that N- and C-terminal Scc1 cleavage fragments remain connected due to their association with different heads of a single Smc1/Smc3 heterodimer. Cleavage of the Smc3 coiled coil is sufficient to trigger cohesin release from chromosomes and loss of sister cohesion, consistent with a topological association with chromatin.
Molecular architecture of SMC proteins and the yeast cohesin complex.
Häring, C.H., Lowe, J., Hochwagen, A. & Nasmyth, K.
Mol Cell. 2002 Apr;9(4):773-88.
Sister chromatids are held together by the multisubunit cohesin complex, which contains two SMC (Smc1 and Smc3) and two non-SMC (Scc1 and Scc3) proteins. The crystal structure of a bacterial SMC "hinge" region along with EM studies and biochemical experiments on yeast Smc1 and Smc3 proteins show that SMC protamers fold up individually into rod-shaped molecules. A 45 nm long intramolecular coiled coil separates the hinge region from the ATPase-containing "head" domain. Smc1 and Smc3 bind to each other via heterotypic interactions between their hinges to form a V-shaped heterodimer. The two heads of the V-shaped dimer are connected by different ends of the cleavable Scc1 subunit. Cohesin therefore forms a large proteinaceous loop within which sister chromatids might be entrapped after DNA replication.
Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion.
Ivanov, D., Schleiffer, A., Eisenhaber, F., Mechtler, K., Häring, C.H. & Nasmyth, K.
Curr Biol. 2002 Feb 19;12(4):323-8.
Cohesion between sister chromatids is established during S phase and maintained through G2 phase until it is resolved in anaphase (for review, see [1-3]). In Saccharomyces cerevisiae, a complex consisting of Scc1, Smc1, Smc3, and Scc3 proteins, called "cohesin," mediates the connection between sister chromatids. The evolutionary conserved yeast protein Eco1 is required for establishment of sister chromatid cohesion during S phase but not for its further maintenance during G2 or M phases or for loading the cohesin complex onto DNA. We address the molecular functions of Eco1 with sensitive sequence analytic techniques, including hidden Markov model domain fragment searches. We found a two-domain architecture with an N-terminal C2H2 Zn finger-like domain and an approximately 150 residue C-terminal domain with an apparent acetyl coenzyme A binding motif (http://mendel.imp.univie.ac.at/SEQUENCES/ECO1/). Biochemical tests confirm that Eco1 has the acetyltransferase activity in vitro. In vitro Eco1 acetylates itself and components of the cohesin complex but not histones. Thus, the establishment of cohesion between sister chromatids appears to be regulated, directly or indirectly, by a specific acetyltransferase.
Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomycespombe.
Häring, C.H., Nakamura, T.M., Baumann, P. & Cech, T.R.
Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6367-72.
The chromosome end-replicating enzyme telomerase is composed of a template-containing RNA subunit, a reverse transcriptase (TERT), and additional proteins. The importance of conserved amino acid residues in Trt1p, the TERT of Schizosaccharomyces pombe, was tested. Mutation to alanine of the proposed catalytic aspartates in reverse transcriptase motifs A and C and of conserved amino acids in motifs 1 and B' resulted in defective growth, progressive loss of telomeric DNA, and loss of detectable telomerase enzymatic activity in vitro. Mutation of the phenylalanine (F) in the conserved FYxTE of telomerase-specific motif T had no phenotype in vivo or in vitro whereas mutation of a conserved amino acid in RT motif 2 had an intermediate effect. In addition to identifying single amino acids of TERT required for telomere maintenance in the fission yeast, this work provides useful tools for S. pombe telomerase research: a functional epitope-tagged version of Trt1p that allows detection of the protein even in crude cellular extracts, and a convenient and robust in vitro enzymatic activity assay based on immunopurification of telomerase.